Masahito Ogasawara

Targeted Disruption of Organic Cation Transporter 3 (Oct3) Ameliorates Ischemic Brain Damage through Modulating Histamine and Regulatory T Cells

The organic cation transporters OCT1, 2, and 3 (SLC22A1-3) have been implicated in the elimination of biogenic amines such as histamine. Among them, OCT3 was identified as an uptake-2 transporter, responsible for clearance of histamine. Because increasing evidence suggests the involvement of histamine in cerebral ischemia, we investigated the effects of targeted disruption of organic cation transporter-3 (Oct3) on the severity of ischemic brain damage. Transient focal ischemia for 1 hour was induced by occlusion of the middle cerebral artery (MCA) of homozygous Oct3-deficient mice and their wild-type (Wt) littermates. Although targeted disruption of Oct3 did not affect physiological parameters after MCA occlusion, this disruption significantly increased histamine content in the ischemic cortex and significantly reduced the infarct volume after cerebral ischemia. Furthermore, targeted disruption of Oct3 prevented the reduction of regulatory T-cell proportion after cerebral ischemia while this disruption did not affect Th1 and Th2 cells proportions after ischemia. Since repeated administration of L-histidine (a precursor of histamine) to Wt mice also showed the same effects, our observations suggested that OCT3 is the molecule responsible for clearance of ischemia-induced histamine in the brain and targeted disruption of Oct3 ameliorated ischemic brain damage through an increase in regulatory T cells.

Lymphangiogenic factors are associated with the severity of hypersensitivity pneumonitis

Background Antigen presenting cells play a pivotal role in the adaptive immune response in hypersensitivity pneumonitis (HP). It was hypothesised that lymphangiogenesis is involved in the pathophysiology of HP via cell transport. Objective To determine the clinical significance of lymphangiogenic factors in HP. Methods Levels of vascular endothelial growth factors (VEGF)-A, VEGF-C, VEGF-D and CCL21 in the serum and bronchoalveolar lavage fluid (BALF) were measured in 29 healthy volunteers, 14 patients with idiopathic pulmonary fibrosis (IPF) and 26 patients with HP by ELISA. Additionally, immunohistochemical analyses were performed using lung specimens of patients with HP (n=8) and IPF (n=10). Results BALF VEGF-D levels were significantly elevated in patients with HP compared to the other groups. BALF VEGF–D levels in patients with HP correlated significantly with the BALF total cell and lymphocyte counts (r=0.485, p=0.014 and r=0.717, p<0.0001, respectively). BALF VEGF-C and CCL21 levels were increased in patients with HP compared to healthy volunteers, but not patients with IPF. BALF CCL21 levels were negatively correlated with the forced expiratory volume in 1 s percentage and diffuse capacity of the lung for carbon monoxide (r=−0.662, p=0.007 and r=−0.671, p=0.024, respectively). According to the immunohistochemical analyses, CCL21 was expressed in the lymphatic endothelium in both conditions and CCR7+ cells were aggregated around lymphatics in patients with HP, but not in patients with IPF. Conclusions Lymphangiogenic factors might be associated with the inflammatory and functional severity of HP. The increased BALF VEGF-D levels were associated with lymphatic alveolitis intensity, and CCL21 with lung function impairment.

Chaperone protein L-isoaspartate (D-aspartyl) O-methyltransferase as a novel predictor of poor prognosis in lung adenocarcinoma.

Endoplasmic reticulum stress and chaperone dysfunction have recently been associated with poor prognoses in various cancers. The newly discovered chaperone protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) regulates the viability of cancer cells in various cancers, although no clinical information regarding the relationship between lung cancer and PIMT expression has been reported. In this study, we aimed to elucidate the relationship between PIMT expression and the prognosis of lung adenocarcinoma. Paraffin-embedded lung tissues obtained from 208 patients with surgically resected lung adenocarcinoma were subjected to immunohistochemical analyses using primary antibodies against PIMT. Kaplan-Meier curves, log-rank tests, and the Cox proportional hazards model were used to analyze the association between PIMT expression and patient survival. Strong PIMT expression was detected in 106 (50.9%) patients, being particularly observed in patients with advanced stages of lung adenocarcinoma. Strong PIMT expression was associated with that of 78-kDa glucose-regulated protein, a marker of endoplasmic reticulum stress. Patients with strong PIMT expression had a shorter survival time (Kaplan-Meier analysis, P<.001). Multivariate Cox hazard regression analysis demonstrated that strong PIMT expression was an independent predictor of poor prognosis of lung adenocarcinoma, including those with stage I disease (hazard ratios, 6.45 and 6.81, respectively; 95% confidence intervals, 2.46-16.9 and 1.79-25.8, respectively; P<.001 and P=.005, respectively). Collectively, strong PIMT expression was a predictive marker of poor prognosis for surgically resected lung adenocarcinoma, and this finding might help clinicians determine the need for postoperative adjuvant chemotherapy in patients with stage I lung adenocarcinoma.

Recombinant Pyrin Domain Protein Attenuates Allergic Inflammation by Suppressing NF-κB Pathway in Asthmatic Mice

Pyrin domain (PYD), a subclass of protein motif known as the death fold, is frequently involved in inflammation and immune responses. PYD modulates nuclear factor‐kappa B (NF‐κB) signalling pathway upon various stimuli. Herein, a novel recombinant pyrin domain protein (RPYD) was generated. Its role and mechanism in inflammatory response in an ovalbumin (OVA) induced asthma model was investigated. After OVA challenge, there was inflammatory cell infiltration in the lung, as well as airway hyper‐responsiveness (AHR) to inhaled methacholine. In addition, eosinophils increased in the bronchoalveolar lavage fluids, alone with the elevated levels of Th‐2 type cytokines [interleukin (IL)‐4, IL‐5 and IL‐13], eotaxin, and adhesion molecules. However, the transnasal administration of RPYD before the OVA challenge significantly inhibited these asthmatic reactions. Moreover, RPYD markedly suppressed NF‐κB translocation, reduced phosphorylation of p38 MAPK, and thus attenuated the expression of intercellular adhesion molecule 1 and IL‐6 in the BEAS‐2B cells stimulated by proinflammatory cytokines in vitro. These findings indicate that RPYD can protect asthma host from OVA‐induced airway inflammation and AHR via down‐regulation of NF‐κB and p38 MAPK activities. RPYD may be used as a potential medicine for the treatment of asthma in clinic.

Deficiency of protein-L-isoaspartate (D-aspartate) O -methyl-transferase expression under endoplasmic reticulum stress promotes epithelial mesenchymal transition in lung adenocarcinoma

A prognostic association between the novel chaperone protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) and lung adenocarcinoma has recently been reported. Here, we evaluated the functional roles of PIMT in the progression of lung adenocarcinoma. PIMT expression was detectable in 6 lung adenocarcinoma cell lines: A549, H441, H460, H1650, Calu 1, and Calu 6 cell lines. In A549 and H441 cells, knockdown by PIMT using silencing RNA of PIMT (si-PIMT) and/or small hairpin-RNA (sh-PIMT) induced a decrease in the expression of E-cadherin with an increase in vimentin expression, indicating that the epithelial to mesenchymal transition (EMT) was induced. Cell mobility, including migration and invasion capability, was increased in sh-PIMT A549 stable and si-PIMT H441 cells compared to in control cells. Endoplasmic reticulum (ER) stress, such as Thapsigargin (Tg) stress and hypoxia, induced EMT in A549 cells but not in other cell types, with an increase in GRP78 expression, whereas overexpression of PIMT reduced the EMT and cell invasion under stress conditions. The expression of hypoxia inducible factor-1 alpha (HIF1α) and Twist increased in sh-PIMT A549 and si-PIMT H441 cells, and Tg stress increased HIF1α expression levels in A549 cells in a dose-dependent manner. Moreover, LW6, an HIF1α inhibitor, reduced EMT, cancer invasion, and the levels of Twist in sh-PIMT A549 cells. Our results indicate that deficiency of supplemental PIMT expression under ER stress facilitates EMT and cell invasion in some cell types of lung adenocarcinoma.

Fractional exhaled nitric oxide levels as a predictor of long-term prognoses in patients with mild asthma

BACKGROUND There is a growing belief that patients with bronchial asthma (BA) should be provided an individualized and optimized treatment plan. We aimed to clarify the predictors of long-term prognoses in patients with mild BA. METHODS We conducted a retrospective study of consecutive patients who were newly diagnosed with mild BA at Iwate Medical University from 2011 to 2013, focusing on achievement of full asthma control based on the Asthma Control Test as an indicator of prognosis. Predictors were identified on the basis of a chart review. RESULTS Among 71 patients with mild BA, 37 patients completed regular clinic visits for 1 year. Nineteen (51.4%) of these patients achieved full asthma control. Current smoking and the fractional exhaled nitric oxide (FeNO) level at the first patient visit were identified by multivariate logistic regression as possible predictors of the discontinuation of clinic visits and achievement of full asthma control, respectively. Low FeNO levels at the first clinic visit yielded a receiver operating characteristic-area under the curve of 0.860 (95% confidence interval [CI]=0.774-0.975) for the achievement of full asthma control. Using an FeNO cut-off level of 34 parts per billion yielded a sensitivity of 76.5% (95% CI=59.5-88.2%) and specificity of 73.7% (95% CI=58.5-84.2%). CONCLUSION Our preliminary results suggested that patients with newly diagnosed mild BA who display higher FeNO levels at their first clinic visits should be appropriately educated during early visits to receive optimal treatment and complete regular clinic visits.

The protective role of protein L-isoaspartyl (D-aspartate) O-methyltransferase for maintenance of mitochondrial morphology in A549 cell.

ABSTRACT Purpose: The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells. Materials and Methods: We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1. Results: We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content. Conclusions: The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.

Galectin-7 as a Marker of Cholesteatoma Residue and Its Detection During Surgery by an Immunofluorescent Method-A Preliminary Study

Objectives To visualize the distribution of galectin-7 in middle ear cholesteatomas using an immunofluorescent method and to establish whether galectin-7 can be used as a marker of cholesteatoma residue at the time of operation. Methods Middle ear cholesteatomas were obtained at surgery from 30 patients. Samples were frozen and preserved in a freezer until histological study. After serial sectioning with a cryostat, 2 of the specimens were processed with primary antibody and Zenon rabbit immunoglobulin G labeling kits. After sufficient reaction time, the samples were observed using a confocal laser microscope. In the remaining 28 specimens, the cholesteatoma was treated as 1 block and stained with the same solution. It was then observed using a fluorescent stereomicroscope. Results Confocal microscopic analyses showed that galectin-7 was distributed in the cholesteatoma matrix. Because this area strongly stained green, it was easily recognized using a confocal laser microscope. In the stereomicroscopic study using the 1-block specimen in which the cholesteatoma was processed together with the surrounding granulation and mucosal tissue, only the matrix and overlying debris was yellow-green in response to excitation by light; the surrounding granulation and mucosal tissues did not respond in 7 specimens. In the remaining 21 specimens, the whole sample was composed of cholesteatoma and responded well to excitation by light. These findings suggest that galectin-7 might be a useful marker of cholesteatoma residue that can be visualized using this immunofluorescent method. Conclusion Because residual cholesteatoma matrix is considered to be one of the main causes of cholesteatoma recurrence, staining with galectin-7 at the time of operation would be a promising way to facilitate complete removal of the residue.